Project Summary/Abstract Nevirapine (NVP) is globally effective in combination treatment of HIV. Its current use in first line therapy is limited by severe immune-mediated adverse drug reactions (IM-ADRs) that significantly adversely impact patient outcome and healthcare costs. Distinct clinical phenotypes of NVP IM-ADRs include Stevens-Johnson Syndrome/Toxic epidermal necrolysis (SJS/TEN) and Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS). DRESS is dependent on both CD4+ and CD8+ T cells and is characterized by fever, rash, hepatitis and eosinophilia. SJS/TEN is a human leukocyte antigen (HLA) class I restricted, CD8+ T-cell dependent hypersensitivity syndrome, which presents as a severe blistering skin rash that can lead to extensive and life-threatening epidermal necrosis. SJS/TEN is the severest IM-ADR and is complicated by a 50% age-dependent mortality rate and severe long-term morbidity in survivors. Pure T-cell mediated ADRs are strongly associated with genetic variation in HLA loci which has led to successful preventive genetic screening such as HLA-B*15:02 screening to prevent carbamazepine SJS/TEN. Current information on key risk HLA alleles specifically associated with NVP IM-ADRs across diverse populations in Africa and Asia is lacking. We have recently identified HLA-C*04:01 as a significant class I risk allele in South African HIV+ patients with NVP SJS/TEN. Another key unanswered question central to the mechanism of HLA-associated IM-ADRs is why hypersensitivity generally occurs in only a small proportion of those carrying an HLA-risk allele. We hypothesize that the immunophenotypic characterization and T-cell receptor (TCR) specificities of the drug- specific T cells in NVP IM-ADR patients will help answer this question. To facilitate the study of IM-ADRs, our research group has established global collaborations that have led to accrual of one of the largest international biobanks of PBMCs, blister fluid and skin biopsies from patients with T-cell mediated ADRs. In Aim 1, I will immunophenotype the specific T cells responsible for NVP SJS/TEN in patients carrying a defined risk allele. I will characterize these NVP-responsive cells based on their cytokine outputs and cell surface and activation markers using enzyme-linked immunospot (ELISpot) assays and flow cytometry respectively. In Aim 2, I will use ELISpot and flow cytometry to characterize the NVP-responsive T cells implicated in NVP DRESS. Finally in Aim 3, I will use single cell TCR sequencing, a Jurkat TCR reporter assay and droplet digital PCR, to identify and confirm the TCR specificities of the T-cell repertoire in patients with NVP IM-ADRs. Our proposed work will leverage clinical and immunologic approaches to define the immunopathogenesis of two distinct cutaneous NVP IM-ADR phenotypes. It is expected that these approaches will ultimately lead to translational outputs such as a sensitive predictive assay to identify the dominant cross-reactive TCRs in the peripheral blood of patients who are destined to develop NVP IM-ADRs. This research will provide the foundation to study the immunopathogenesis of other IM-ADRs and provide a roadmap for their preclinical prediction and prevention.